Genetic Transformation of Artemisia by rot genes

Abstract

Artemisinin, a potent antimalarial drug is the major constituent of Artemisia species. The aim of present study was to compare artemisinin production in the roots of Artemisia dubia transformed with rol ABC genes by using Agrobacterium tumefaciens with the non transformed plants. As a number of factors have been reported that affect the efficiency of transformation with Agrobacterium tumefacienes, different parameters li ke type of explants, sterilization condition and co-cultivation period. Agrobacleriul'l1 tumefacienes strain LBA4404 containing pRT99 harboring rol ABC genes were used [or the transformation experiments. Mercuric chloride 0.1 % for 2 minutes showed the best results for seed surface sterilization with 95% germination. Leaves and stems were found to be the best explants for transformation. Dipping explants in bacterial suspension for 10 minutes and co-cultivating them on selection medium eMS medium with 200 ~l M acetosyringone and 50mg/l kanamycin) for 48 hours resulted in the maximum number oC transformants. Regeneration of transgenic shoots was obtained from both stem and leaves explants on regeneration medium eMS medium containing 1.0mg II BAP supplemented with 20mg/ml kanamycin and 00~lg ml cefotaxime). Eighty percent of the transgenic shoots showed rooting response on Half MS medium with 0.025 mg/l NAA. Control and the transgenic plants were transferred to small pots containing soil and acclimatil'.ed. Morphological differences like increased plant height and broad leaves were observed. Transformed plants were selected on 100ug/ml kanamycin and final selection of transformed plants was made on the basis of PCR for the detection of rol ABC genes. Transformed and control roots were subcultured on solid B5 medium for further root proliferation. Better roots proliferation was observed on solid B5 medium in the case of transformed root while control root showed minimum proliferation. After 30 days of roots proliferation Artemisinin content was analyzed with the help of spectrophotometer. Maximum A11emisinin was observed in transformed roots of Artemisia dubia while minimum content of Artemisinin was observed in control plants.